SPring-8, the large synchrotron radiation facility

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Successful tracking of single biomolecule motion with extremely low-dose X-ray (Press Release)

Release Date
30 Nov, 2018
  • BL40B2 (Structural Biology II)
-Acceleration of development of ultrahigh-sensitivity detection method and expansion of its application-

November 30, 2018
The University of Tokyo
National Institute of Advanced Industrial Science and Technology
Japan Agency for Medical Research and Development
Japan Synchrotron Radiation Research Institute

Key points
◆ We succeeded in time-resolved tracking of the ultrafine motion of gold nanoparticles labeled on a single molecule using monochromatic X-rays (Note 1) generated at a large synchrotron radiation facility (SPring-8) and a laboratory-scale small X-ray source.
◆ Compared with conventional diffracted X-ray tracking (DXT), the new method enables tracking of molecular motion with a 1/1700-fold X-ray dose (Note 2), meaning that a laboratory-scale small X-ray source can be used.
◆ Because the damage caused by X-rays is reduced, molecular motion in living cells and animals can be observed over a long period of time.

Recently, techniques for observing a single protein molecule have rapidly advanced, enabling in vivo observation of molecular dynamics with high speed and sensitivity. In the conventional DXT method (Note 3), the specific site of a target protein is labeled with gold nanocrystal and the trajectory of X-ray diffraction spots from the gold nanocrystal is tracked to observe the internal motion of a protein molecule with a high time resolution, such as less than 1 μs, and a high sensitivity of picometer order. Researchers of the University of Tokyo have successfully tracked the internal motion of a single protein molecule of deoxyribonucleic acid (DNA) and a giant membrane protein by the DXT method.

The research group of Yuji Sasaki (professor, Graduate School of Frontier Sciences of the University of Tokyo, also affiliated with AIST-UTokyo Advanced Operando-Measurement Technology Open Innovation Laboratory) and a research group of Japan Synchrotron Radiation Research Institute (JASRI) have jointly observed, for the first time in the world, the blinking phenomenon of the intensity of X-ray diffraction (blinking X-ray, Note 4) caused by the motion of gold nanocrystals labelled with the target molecule. In the diffraction experiment, monochromatic X-rays from beamline BL40B2 at SPring-8 (Note 5) were used. They demonstrated that the motion of the diffraction X-ray spots can be quantitatively evaluated by analyzing the autocorrelation (Note 6) function of diffraction intensities. In addition, it was found that the X-ray dose required for the observation of diffraction X-ray blinking is 1/1700 that in the conventional DXT method, which, up to now, had been the only method for tracking the internal motion of a single molecule. In other words, with this new method of using monochromatic X-rays, tracking the motion of a single molecule is realized with an extremely low dose of X-rays. In addition, taking advantage of the low dose, the dynamics of a single molecule was tracked using a laboratory-scale X-ray source at a time resolution of millisecond order.

Publication: Scientific Reports 8, Article number: 17090 (2018)
Title: Diffracted X-ray Blinking Tracks Single Protein Motions
Authors: H. Sekiguchi2, M. Kuramochi1, K. Ikezaki1, Y. Okamura1, K. Yoshimura1, K. Matsubara1, J. W. Chang1, N. Ohta2, T. Kubo3, K. Mio3, Y. Suzuki1, L. M. G. Chavas4 and Y. C. Sasaki1, 2, 3, (Corresponding author: H. Sekiguchi, Y. C. Sasaki)
DOI: 10.1038/s41598-018-35468-3
1 Graduate School of Frontier Sciences of the University of Tokyo, 2 Japan Synchrotron Radiation Research Institute (JASRI/SPring-8), 3 AIST-UTokyo Advanced Operando-Measurement Technology Open Innovation Laboratory, 4 Proxima-I, Synchrotron SOLEIL


(Note 1) Monochromatic X-rays from synchrotron radiation
Synchrotron radiation is a narrow and powerful electromagnetic wave (light) generated when electrons are accelerated to a speed comparable to that of light and their path is bent by an electromagnet. The generation of continuous X-rays of various wavelengths is possible. Continuous X-rays irradiated on a monocrystal (diffraction grating) are reflected (diffracted). The angle of diffraction depends on Bragg’s law and only waves with a specific wavelength can be reflected. In other words, only waves with a specific wavelength can be collected by diffraction from continuous X-rays of various wavelengths. The element used for this purpose is generally called a monochromator. In the X-ray wavelength range, silicon monocrystals are often used as the monochromator material. Monochromatic X-rays obtained using the monochromator are used in measurements employing the beamlines of general synchrotron radiation facilities.

(Note 2) X-ray dose
X-ray dose is the amount of radiation absorbed upon X-ray irradiation. At medical sites, X-ray dose is used as the unit of radiation absorbed by the organs of patients, because it is independent of the kind of irradiated material. An X-ray probe is sometimes called a nondestructive probe because the damage to the sample is extremely small compared with that of an electron beam, which is also a quantum probe. However, we cannot ignore the destruction of a sample caused by the photoelectric effect when the sample is irradiated with high–intensity high-energy X-rays. A countermeasure to this problem has been sought.

(Note 3) Diffracted X-ray tracking (DXT) method
In the DXT method, the molecules or aggregates that are the target of dynamics evaluation are labeled with gold nanocrystals having a diameter of several nanometers; then, the motion of X-ray Laue spots diffracted from the gold nanocrystals is observed with a high-speed time resolution. The DXT method mainly detects the rotational motion with a sensitivity of less than milliradian order (0.01o), which is equivalent to a translation distance of picometer order. The time resolution is as fast as 100 ns, although it depends on the performance of the high-speed camera used. The above values are the fastest reported for tracking a single molecule in the visible range. This technique was developed by Professor Yuji Sasaki in 1998 and published in 2000. His research group has already published many reports on the internal motion of a protein molecule (e.g., Physical Review Letters, Physical Review, BBRC, Cell, Biophysical J., and Scientific Reports).

The principle of the DXT method is shown in the figure below. It is the only method that detects the internal rotational motion of dynamic molecular aggregates (nonuniform structural materials). They named this new method in which DXT is realized using monochromatic X-rays the diffracted X-ray blinking (DXB) method. The mechanisms behind DXT and DXB are shown in the figure.

Diffracted X-ray tracking (DXT) method

(Note 4) Diffraction X-ray blinking (DXB) method
In the 1990s, the blinking phenomenon at intervals of the order of milliseconds to seconds was observed in the visible light region. It was considered to be a typical phenomenon to be avoided because it made measurements unstable. The blinking phenomenon has been observed for various pigments used for the tracking of a single molecule, including green fluorescent protein (GFP), organic fluorescent molecules, and semiconductor quantum dots. Professor W. E. Moerner of Stanford University excited yellow fluorescent protein (YFP), a variant of GFP, with light of a wavelength of 488 nm and observed the luminescence property of a single molecule of YFP by a special optical microscopy method. He found that the fluorescence began to blink. He further found that after several blinks, it eventually stopped and the YFP became stable. He was awarded the Nobel Prize in Chemistry for this achievement in 2014. The study led by Professor Sasaki and his colleagues was the first instance of observing the blinking of X-ray diffraction intensity, as shown in the figure. It was also confirmed that the phenomenon is independent of the type of X-ray source and originates from the motion of the nanocrystals used as the X-ray diffraction source.

Diffraction X-ray blinking (DXB) method

(Note 5) Large synchrotron radiation facility (SPring-8)
SPring-8, owned by RIKEN, is a large synchrotron radiation facility (public interest incorporated foundation) that delivers the most powerful synchrotron radiation in the world and is located in Harima Science Garden City in Hyogo Prefecture, Japan. JASRI operates and supports users of the facility. The name “SPring-8” is derived from “Super Photon ring-8 GeV”. The synchrotron radiation is in the form of a narrow and powerful beam of electromagnetic light generated when electrons are accelerated to a speed comparable to that of light and their path is bent by an electromagnet. The research conducted at SPring-8 covers a wide range of fields from nanotechnology to biotechnology to industrial applications.

(Note 6) Autocorrelation
Autocorrelation is a mathematical method of data analysis that is based on a time axis. Autocorrelation is the correlation of a signal with a delayed copy of itself and is represented as a function of the time shift. Autocorrelation is effective in revealing minute patterns included in signals. It is a fundamental primary analysis method that enables more detailed analysis using the results of the primary analysis. Autocorrelation is used in single-particle analysis by electron microscopy and dynamic optical scattering in the visible light region. In the X-ray region, X-ray photon correlation spectroscopy has been used, although the measurement conditions are limited and only special samples can be measured. In contrast, the DXB method developed and demonstrated in this study is expected to be a general-purpose method because it accommodates a wide range of measurement conditions.

Concept of DXB method and comparison of DXT and DXB measurements are summarized as following movie,

Concept of DXB

Comparison of DXT and DXB measurements

Professor Yuji Sasaki
Graduate School of Frontier Sciences of the University of Tokyo

National Institute of Advanced Industrial Science and Technology

Japan Agency for Medical Research and Development

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